Pancreatic cancer is the fourth-leading cause of cancer-related death in the U.S. and remains a largely intractable disease. Nearly all cases of pancreatic ductal adenocarcinoma(PDAC) are characterized by activating mutations in the Kras gene. Attempts at pharmacological inhibition of Kras and its downstream signaling effectors in PDAC have so far been unsuccessful. Cancer cell metabolism has become a new area of interest for the development of targeted therapies. Through activation of various signaling pathways, oncogenic Kras induces a metabolic transformation that makes pancreatic cancer cells more reliant on glycolysis and less reliant on mitochondria-based metabolism. The proposed study focuses on Nix, a protein involved in mitochondrial maintenance and turnover, which might be responsible for the metabolic transformation seen in Kras-mutant cells. Our preliminary studies show that Nix is upregulated at the RNA and protein levels in murine pancreatic tumors. Additionally, ablation of Nix or Kras leads to a decrease in the number of mitochondria and the proliferative capacity of Kras-mutant, but not Kras wild-type, cancer cells. These results suggest the exciting possibility that Nix might be a legitimate drug target for the treatment of Kras-mutant pancreatic cancer. The proposed study will further elucidate the role of Nix in pancreatic cancer using genetically engineered mouse models and a novel pancreatic ductal organoid culture system. First, we will evaluate the metabolic consequences of Nix ablation using the organoid system. For the first time in vitro, this system will allow us to compare the metabolic phenotype of normal, pre-neoplastic and neoplastic pancreatic cells expressing or lacking Nix. Second, we will determine the role of Nix in pancreatic tumor intiation and maintenance using genetically engineered mouse models and an orthotopically transplanted organoid mouse model (respectively). We have crossed mice expressing a conditional Nix allele into our KPC mouse model, characterized by pancreas- specific expression of mutant Kras and mutant p53, to study the effects of Nix on pancreatic tumorigenesis. To study PDAC maintenance, we will transplant organoids infected with inducible short hairpin RNAs (shRNAs) targeting Nix into the pancreata of wildtype mice. These shRNAs will be induced after tumor formation in vivo to assess the effects of Nix knockdown on tumor maintenance. Results from these studies will not only validate the legitimacy of Nix as a suitable drug target for pancreatic cancer, but will also elucidate the role of Nix in the metaboli transformation of pancreatic cancer to provide a better understanding of this therapeutic target.